Method of identifying proteins modified by disease states related thereto

ABSTRACT

A new class of polypeptides is disclosed, along with a method for identifying and producing such polypeptides, having the characteristic of being unique to diseased states, particularly tumors and blood-borne malignancies. These new polypeptides are active and will be useful for therapeutic purposes.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 08/257,784filed Jun. 10, 1994, now U.S. Pat. No. 5,789,556, which is acontinuation in part of U.S. application Ser. No. 08/076,231, filed Jun.11, 1993 now abandoned.

FIELD OF THE INVENTION

This invention relates to the field of biotechnology and moreparticularly to polypeptides having antitumor, antiviral,immunomodulatory and other activities, DNA that codes for suchpolypeptides, recombinant vectors that include such DNA, host organismstransformed with the recombinant vector that produces the polypeptideand therapeutic application of the polypeptide.

The publications and other materials hereof used to illuminate thebackground of the invention, and in particular cases, to provideadditional details respecting its practice are hereby incorporated byreference, and for convenience are numerically referenced by thefollowing text and respectively grouped in the appended bibliography.

BACKGROUND OF THE INVENTION

Human leukocyte interferon was first discovered and prepared in the formof very crude fractions by Isaacs and Lindenmann (1, 2). Efforts topurify and characterize the material have led to the preparation ofrelatively homogeneous leukocyte interferons derived from normal orleukemic (chronic myelogenous leukemia or "CML") donors leukocytes.These interferons are a family of proteins characterized by a potentability to confer a virus-resistant state in their target cells. Inaddition, interferon can inhibit cell proliferation, modulate immuneresponses and alter expression of proteins. These properties haveprompted the clinical use of leukocyte interferon as a therapeutic agentfor the treatment of viral infections and malignancies.

With the advent of recombinant DNA technology, the controlled microbialproduction of an enormous variety of useful polypeptides has becomepossible. The workhorse of recombinant DNA technology is the plasmid, anon-chromosomal circle of double-stranded DNA found in bacteria andother microbes, oftentimes in multiple copies per cell. Included in theinformation encoded in the plasmid DNA is that required to reproduce theplasmid (i.e. an origin of replication) and ordinarily, one or moreselection characteristics such as, in the case of bacteria, resistanceto antibiotics which permit clones of the host cell containing theplasmid of interest to be recognized and preferentially grown inselective media. The utility of plasmids lies in the fact that they canbe specifically cleaved by one or another restriction endonuclease or"restriction enzyme", each of which recognizes a specific site in theDNA. Heterologous genes or gene fragments may be inserted into theplasmid at the cleavage site. To construct vectors with specificsequences inserted, DNA recombination is performed outside the cell, butthe resulting "recombinant" plasmid can be introduced into cells by aprocess known as transformation and large quantities of the heterologousgene-containing recombinant plasmid obtained by growing thetransformant. Moreover, where a promoter which governs the transcriptionof the encoded DNA message, is properly placed upstream (5') of a codingsequence or a gene, the resulting expression vector can be used toproduce the polypeptide sequence for which the inserted sequence or genecodes, a process referred to as expression.

Expression is initiated in a region known as the promoter which isrecognized by and bound by RNA polymerase. In many cases promoterregions are overlapped by "control" regions such as the bacterialoperators. Operators are DNA sequences which are recognized by so-calledrepressor proteins which serve to regulate the frequency oftranscription initiation at a particular promoter. The polymerasetravels along the DNA, transcribing the information contained in thecoding strand from its 5' to 3' end into messenger RNA (mRNA) which isin turn translated into a polypeptide having the amino acid sequence forwhich the DNA codes. Each amino acid is encoded by a nucleotide tripletor "codon" within the coding sequence, i.e., that part which encodes theamino acid sequence of the expressed product. In bacteria (e.g.Escherichia coli) the mRNA contains a ribosome binding site, atranslation initiation or "start" signal (ordinarily ATG in the DNA,which in the resulting mRNA becomes AUG), the nucleotide codons withinthe coding sequence itself, one or more stop codons, and an additionalsequence of messenger RNA, the 3' untranslated region. Ribosomes bind tothe binding site provided on the messenger RNA, in bacteria ordinarilyas the mRNA is formed, and produce the encoded polypeptide, beginning atthe translation start signal and ending at the stop signal. The desiredproduct is produced if the sequences encoding the ribosome binding siteare positioned properly with respect to the AUG initiator codon and ifall remaining codons follow the initiator codon in phase. The resultingproduct may be obtained from the host cell and recovered by appropriatepurification. In other systems, proteins may be secreted from the hostcells. A wide variety of expression vectors and host systems exist sothat RNA and proteins may be expressed in prokaryotic and eukaryoticcells as well as in intact animals and plants.

During the past several decades a large number of human and animalinterferons have been produced, identified, purified and cloned (seeref. 1-72). Several of the interferon preparations have been preparedfor clinical trial in both crude form, for some of the originalinterferon preparations, as well as in purified form. Several individualrecombinant interferon-α species have been cloned and expressed. Theproteins have then been purified by various procedures and formulatedfor clinical use in a variety of formulations (73). Most of theinterferons in clinical use that have been approved by variousregulatory agencies throughout the world are mixtures or individualspecies of human α interferon (Hu-IFN-α). In some countries Hu-IFN-β andγ have also been approved for clinical trial and in some cases approvedfor therapeutic use (56, 74). The major thesis underlying clinical useof these interferons was that they were natural molecules produced bynormal individuals. Indeed, the specific thesis was that all theinterferons prepared for clinical use, by they natural- orrecombinant-generated products, represented interferons that wereproduced naturally by normal people. This is true for a large number ofinterferons as well as specific growth factors, lymphokines, cytokines,hormones, clotting factors and other proteins that have been produced(17, 21, 22, 25-27, 29-34, 39, 40, 45-51, 53-57, 62-64, 68-72).

Reports have suggested that Hu-IFN-αA (also designated Hu-IFN-α2α and bythe trade name Roferon A) was not represented in interferons produced bya normal population of individuals (75-79). Believing that certaininterferons (or, more generically, certain polypeptides) are uniquelyfound in diseased cells, the inventor of the present invention undertookto identify interferons which are so uniquely characterized. Forconvenience the inventor began by screening known interferons, inparticular, the sources of the several variants of Hu-IFN-α2 that havebeen described. As discussed more fully below, it was found that thesource of two of the variants of Hu-IFN-α2, Hu-IFN-α2a and Hu-IFN-α2c,are not present in normal individuals. Only Hu-IFN-α2b is found innormal individuals (79).

DESCRIPTION OF THE DRAWINGS

FIG. 1 Nucleotide and Amino Acid Sequence of Hu-IFN-α001 (SEQ ID NOS:1and 2). The location of the AlwNI site is underlined. The signal peptideis shown as the 23 amino acids labeled -1 to -23.

FIG. 2 Comparison of the Protein Sequence of Hu-IFN-α001 (SEQ ID NO:2)with that of Hu-IFN-αJ (SEQ ID NO:3). The signal peptide represents thefirst 23 amino acids at the amino terminus.

FIG. 3 Expression vector for Hu-IFN-α001. The structure of the plasmidpHu-IFN-α001 is shown. The NsiI site represents nucleotide position #1.The P_(R) promoter drives expression of Hu-IFN-α001.

FIG. 4 SODS-Polyacrylamide Gel Electrophoresis of the PurifiedHu-IFN-α001, Hu-IFN-α001 was placed in lanes 1, 2 and 3 in amounts of 3μg, 1.5 μg and 0.75 μg, respectively. The columns labeled M representthe molecular weight markers with the values in kilodaltons given to theleft of each respective molecular weight marker.

SUMMARY OF THE INVENTION

An extensive analysis of normal individuals from various ethnic andracial backgrounds as well as two tumor cell lines has shown thatcertain interferons originated from two cell lines that were obtainedfrom patients with disease, in particular, malignancies of thehemopoietic system (79). These results lead the inventor to concludethat there is a new class of interferon molecules which are present indiseased states, specifically in tumors and blood borne malignancies.This discovery of a new class of interferons provides a wide variety ofpotentially new interferons for clinical and therapeutic use. Theseinterferons include not only Hu-IFN-α species, but also Hu-IFN-β,Hu-IFN-γ and Hu-IFN-ω, as well as other newly described interferons inother animals and species. The observations suggest that growth factors,cytokines, lymphokines, clotting factors, peptide and polypeptidehormones, adhesion factors and many other molecules are also modified indisease processes. Therefore, modified forms of all these cytokines,lymphokines, growth factors, adhesion molecules, enzymes, clottingfactors, peptide and polypeptide hormones, etc. will also occur intumors and other diseases. Based on two presently identified members ofthis class (not previously recognized as such), these interferons areactive, are as active as the standard molecules, and in fact have beenused effectively for therapeutic purposes. A paper co-authored by theinventor and listed hereafter as Reference No. 79 is particularlyrelated to the invention and is incorporated herein by reference insofaras may be needed for a full understanding of the invention. That paperis more fully described, and is furnished as an attachment to acontemporaneously filed Information Disclosure Statement.

DETAILED DESCRIPTION OF THE INVENTION

Four distinct classes of interferons (IFNs) are known to exist inhumans. The IFN-α family represents the predominant class of IFNs andare produced by stimulated peripheral blood leukocytes (10-15, 17-27,29, 50, 51, 57-59, 61, 63, 64, 68, 70), and lymphoblastoid andmyeloblastoid cell lines (28, 30, 60). Cloning of the IFN-α genes fromthese cells has revealed that IFN-α is encoded by a multigene familyconsisting of about 15 functional genes and four pseudogenes (17, 26,27, 29, 31, 50, 51, 53, 54, 57, 61, 63, 64, 65). It has been uncertainwhether or not some of the cloned human IFN-α genes and cDNAs with fewnucleotide differences, such as the Hu-IFN-α-A. Hu-IFN-α2 andHu-IFN-α2(Arg) genes, are allelic variants or represent distinct genes.

To determine if these sequences do indeed represent separate genes orare instead polymorphic variants of a single gene, sequencesrepresenting only the Hu-IFN-αA, Hu-IFN-α2 and Hu-IFN-α2(Arg) genes wereamplified by nested polymerase chain reaction (PCR) from human genomicDNAs of healthy consenting individuals. These sequences were thensubcloned and examined by sequencing of individual clones. In addition,the DNAs were examined from KG-1 (80) and Namalwa (81) cell lines fromwhich the Hu-IFN-αA and Hu-IFN-α2(Arg) cDNAs, respectively, were cloned.

MODES FOR CARRYING OUT INVENTION

Three oligodeoxynucleotides were prepared by the phosphoramidite method(82, 83) and purified (84). Primer I(5'-TGGGCTGTGATCTGCCTC-3') (SEQ IDNO:4) complementary to nucleotides 125 to 142 at the 5' end was usedwith Primer II (5'-CATGATTTCTGCTCTGACAACC-3') (SEQ ID NO:5))complementary to nucleotides 552 to 573 at the 3' end to amplify thedesired nucleotide sequences. The DNA, as amplified by the polymerasechain reaction (PCR) with this primer pair, was expected to representsequences from most of the IFN-α gene family (79). This conserved PCRproduct was then used as template in a second amplification reactionwith the same 3' oligonucleotide but with a 5' oligonucleotide specificfor the human IFN-αA, IFN-α2 and IFN-α2(Arg) genes only (79). The secondreaction produced a product of 430 bp when PrimerIII(5'-AACCCACAGCCTGGGTAG-3') (SEQ ID NO:6) complementary to nucleotides144 to 161 was substituted for the Primer I. The 430 by DNA was purifiedand cloned into the SmaI site of pBluescript-SK⁻ (Stragene, LaJolla,Calif.) as described (79, 85, 86).

DNA of the plasmids was prepared by the alkaline lysis miniprepprocedure (86, 87) from 1 ml cultures grown overnight in LB mediumcontaining 100 μg/ml ampicillin. The resultant DNA pellet was sequencedby the dideoxy chain termination procedure (79, 88, 89). The reactionswere run on 6% polyacrylamide gels which were then dried and and exposedto X-ray film overnight at room temperature with an intensifying screen.

Reverse transcriptase PCR (RT-PCR) was used to analyze the expression ofthe IFN-α subtypes αA, α2 and α2(Arg) in the KG-1 and Namalwa cell lines(90). RNA was isolated at 6 hours after induction from Sendaivirus-induced KG-1 cells (60) and at 8 hours post induction fromNDV-stimulated Namalwa cells (91, 92).

DNA was extracted from the human myeloblastoid cell line KG-1 and fromthe lymphoblastoid Namalwa cell line by a modification of the method ofPellicer et al. (93). After obtaining informed consent, human genomicDNA was prepared from whole blood samples collected from normal, healthyindividuals by ammonium acetate precipitation as described (79, 94).

METHODOLOGICAL BASIS FOR INVENTION

The DNA from 11 normal individuals was amplified by nested PCR thencloned and sequenced as described above. The number of sequencescorresponding to the various human IFN-α species is shown in Table 1. Itcan be seen that neither the sequence for the αA gene nor the α2(Arg)gene was detected in any of the normal individuals examined in thisstudy. As shown in Table 2, however, the αA sequence was detected in theDNA from the KG-1 cell line, but not in Namalwa cells; and the α2(Arg)sequence was detected in the DNA from the Namalwa cell line, but not inKG-1 cells.

                  TABLE 1                                                         ______________________________________                                        Frequency of Hu-IFN-αA, -α2 and α2(Arg) Clones From         Normal                                                                          Individuals                                                                        Interferon Variant                                                                        Number of Clones                                           ______________________________________                                        IFN-α2   165                                                              IFN-αA 0                                                                IFN-α2(Arg) 0                                                           Other.sup.1 36                                                                Total 201                                                                   ______________________________________                                         .sup.1 Other refers to sequences which contained one or more mutations in     an area unrelated to the αA and α2(Arg) specific differences.     It should be noted that the frequency of mutations detected is in the         range or slightly lower than that predicted from the combined error rates     of Taq DNA polymerase and Sequenase DNA polymerase (95, 96). Previous         analysis of IFNα2 genes have been reported (97.98). but did not         discern any differences in their representation                               # in the DNA from normal individuals. Descriptions and abbreviations          relevant to interferons are described in detail in several references         (10-12, 61, 99, 100).                                                    

                  TABLE 2                                                         ______________________________________                                        Frequency of IFN-α Clones From KG-1 and Namalwa Cell Lines                Cells      IFN-α2                                                                            IFN-αA                                                                        IFN-α2(Arg)                                                                      Other                                                                              Total                              ______________________________________                                        KG-1 cells                                                                             15        10      0        16   41                                     Namalwa cells 22 0 13 2 37                                                  ______________________________________                                    

Restriction endonuclease analysis to detect the IFN-αA gene was alsoperformed on DNA from five of the individuals from whom clones had beensequenced and on DNA from seven additional people that were not examinedby DNA sequencing. It was found that the restriction endonucleaseanalysis of the amplified DNA from all of these individuals showed noIFN-αA gene present (See Ref. 79, FIG. 2).

PREFERRED EMBODIMENT OF INVENTION

From the foregoing analysis, it can be concluded that in human DNA froma wide variety of humans only Hu-IFN-α2 is present. The speciesHu-IFN-αA and Hu-IFN-α2(Arg), not present in the DNA of 11 normalindividuals, apparently arose during the development of the diseaseand/or the establishment of the cell lines in culture. It is noteworthythat the expression of these alleles of Hu-IFN-α2 yields IFN-α specieswith high activity in a wide variety of assays (63, 68, 69, 101-115).The specific activities of all three of these IFN-α species arecomparable. Furthermore, it has been reported that patients treated withHu-IFN-αA produced a higher level of anti-interferon antibodies thanpatients treated with Hu-IFN-α2 or HU-IFN-αn (Welferon: a preparation ofmixed Hu-IFN-α species produced by induced Namalwa cells) (116-124).Some of the new interferons produced by the described invention may beable to by-pass neutralization by the antibodies produced in patientstreated with IFN-α preparations in current use. Such new IFN-α speciesshould be able to be used to treat patients who have relapsed because ofneutralization of the administered IFN-α species.

While the inventor has, for convenience, used Hu-IFN-α2 and its knownvariants for establishing his hypothesis of the existence of a class ofsuper or tumor interferons, it will be apparent to those skilled in theart that the results extend to an entire class of such interferons, aswell as other polypeptides. Illustrative of this conclusion is theextraordinary high percentage of variant forms of the IFN-α2 and αAgenes in KG-1 cells--i.e. 39% (16/41), much more than could be explainedby experimental error, as shown in the column labeled "Other" of Table2.

It will also be apparent that the method of the invention, asillustrated above for Hu-IFN-α2, can be applied to any protein. In thegeneral case, a primer pair is chosen to encompass part or all of thenucleotide coding sequence with the use of DNA from tumor cells or fromcultured cells as templates for the PCR. The PCR product is then clonedand sequenced. The amino acid sequence predicted by the nucleotidesequence so obtained is compared to the sequence of the protein innormal individuals. Proteins with amino acid sequences different fromthose proteins in normal individuals are then cloned in appropriateexpression vectors (11, 12, 14, 17, 45, 53, 54, 57, 63, 69, 86, 103),produced, purified and characterized. Those with desirable activitiesare then developed for therapeutic use.

The origin of the tumor interferons or super interferons is unknown.Yet, it is clear that they are developed during the pathologicalprocess. It is believed that the cells producing these interferons havebeen selected during development and progress of the disease.

The presence of allelic forms of IFN-α2 in the KG-1 and Namalwa cells ismost noteworthy. DNA from leukocytes from normal individuals did notcontain these variants. Because both the KG-1 and Namalwa cellsoriginated from patients with leukemia or lymphoma, it is believed thatthis alteration is an early change in progession of these diseases.Indeed, it has been reported that there are significant gross changes inrestriction endonuclease digestion patterns for the IFN-α genes in acuteleukemias (125, 126).

The disease mechanisms involved in developing malignant cells andselection of those cells produce a wide variety of genetic changes inthe resultant tumor cells. In order for cancer cells to grow unfettered,to escape the normal controls and to metastasize, the usual regulatorynetwork of the immune system, involving growth inhibitors as theinterferons and growth factors and hormones, may be modified. Thecontrol of cell growth and nonmalignant behavior is a delicate balanceof many regulatory factors, a few of which gone astray can alter thenormal growth patterns. Although it has been reported that changes inthe DNA of cancer cells occurs, the changes have been focussed ononcogenes and tumor suppressor genes that lead to the malignantphenotype. The inventor has provided data that the changes are morepervasive than expected, not merely those changes focussed on oncogenesand tumor suppressors. Furthermore, by genetic changes (mutations inDNA) and selection of tumor cells for aggressiveness, many alterationswill be embodied in the final tumor cell population. The new proteinsproduced will have lower, the same or higher activities than the normalproteins. By identifying those modified proteins associated with changesin activity, it will be possible to identify those proteins with newand/or enhanced activities.

From an analysis of initial clones obtained from the KG1 cell line (53,54, 63), is was shown that several abnormal interferons exist in thiscell line (also see ref. 61 for list of IFN-α species). This isespecially evident in that αB (not previously recognized as an abnormalinterferon) has an insertion and compensating deletion making anabnormal protein that differs from Hu-IFN-α8, the normal counterpart.The presence of an insertion and a compensating deletion producing anormal sized molecule suggests some enormous selective pressures toproduce these interferons. The fact that an insertion and a deletionwould be incorporated into a molecule simply by random stochasticprocesses without external pressures is highly unlikely. Thus, thesemodified interferons are seen to represent an entire new family ofmolecules that have been developed under the pressure of enormousexternal forces to provide for the selection of these species.

With respect to the interferons produced by mechanisms to enhance orcombat the disease process, some may indeed have unusual properties andmay be more active than some of the interferons produced by normalcells. For example, since interferon is a growth inhibitory molecule,production of a new interferon that could down regulate the receptorsfor interferon in cells and help select for cells without interferonreceptors or low levels of interferon receptors may enhance the diseaseprocess. Such interferons could also help select cells with an alteredsignal transduction mechanism, but normal receptor number. Thus, a cellproducing spontaneously some interferon, could be expected to haveinitially a low level of receptors due to down regulation and its growthwould likely be reduced. Nevertheless, during the multiplication of suchcells, cells would be selected that would have low levels of receptorsso that they could escape the inhibition of the endogenous interferon.The same would hold for a wide variety of other molecules such ascytokines, lymphokines, tumor suppressors, growth factors, anti-growthfactors, matrix molecules, hormones, angiogenic factors, clottingfactors, etc., all molecules that can control growth and/or metastasesin one manner or another.

The altered proteins described herein are found in tumor cells orcultured cells obtained from tumors. Furthermore, selection ofcell-lines in culture can also produce some of the alterations asselection in vivo.

CLONING OF Hu-IFN-α001

A new interferon was amplified from the genomic DNA of KG-1 cells (ATCCCCL 246) based on the strategies outlined hereinbefore and by theprocedures described herein and elsewhere (79). The primers used toamplify the genes are shown in Table 3. The 5' primer contains an ApaIsite, and the 3' primer contains an XbaI site for cloning. The PCRreactions were carried out in 50 μl with 100 ng KG-1 template DNA, 100ng of each primer (6431 and 6432), 0.2 mM of each dNTP, and 2.5 units ofTaq DNA Polymerase for 30 cycles of 94° C. 30 seconds. 50° C. 30seconds. 72° C. 30 seconds in the Perkin Elmer model 9600 thermocycler.Products of the PCR amplification were cloned into the ApaI and XbaIsites of plasmid pBluescript II KS+ (Stratagene) and then transformedinto competent E. coli strain DH5α cells. Competent cells were preparedin 12% PEG and 36% glycerol in Luria-Bertani medium (L-broth medium, 10g tryptone, 5 g yeast extract, 10 g NaCl, pH 7.3) from Digene (SilverSpring, Md. 20904, Cat. No. 3500-1002) as described (127). Plasmid DNAwas isolated from 2.0 ml of overnight cultures grown at 37° C. by amodified alkaline lysis procedure as reported (128). The size of theinserts was determined by digestion with restriction endonucleases KpnIand SacI that flanked the cloning sites in the vector pBluescript. Atotal of 10 independent colonies were identified that contained a 700base pair insert.

                  TABLE 3                                                         ______________________________________                                        Primers for PCR Amplification                                                                                        Primer                                   Primer Sequence Length No.                                                  ______________________________________                                        5'     GCGGGCCCCAATGGCCYTGYCCTTT                                                                           25      6431                                        SEQ ID NO:7                                                                  3' GCTCTAGAAYTCATGAAAGYGTGA 24 6432                                            SEQ ID NO:8                                                                ______________________________________                                    

The DNA from one of the clones (plasmid pBS001) was sequenced in bothdirections. Automated DNA sequencing was performed on a Genesis 2000Automated DNA Sequencer (DuPont, Wilmington, Del.) with the primersshown in Table 4 by methods previously reported (86, 88, 89). Allsequences were performed on both strands. Automated sequencing wascarried out and the results were compiled to create a consensussequence. The sequence determined from the T3 primer represents the 5°end of the insert; the T7-derived sequence represents the 3' end.

The sequence so determined is designated Hu-IFN-α001 and is shown inFIG. 1 (SEQ ID NO:1). The location of the AlwNI restriction endonucleaserecognition site (5' CAGNNCTG 3') (SEQ ID NO:9) that was used for thesplicing of the Hu-IFN-α001 insert into the expression vector TGATG(129) is indicated in the figure by underlining. The signal peptide isshown as the 23 amino acids labeled -1 to -23. As seen in FIG. 1 (SEQ IDNO:2), the mature protein contains 166 amino acids.

                  TABLE 4                                                         ______________________________________                                        Primers used for Sequencing                                                                                     Position in                                                                          Direc-                                 Design-  Primer Hu-IFN- tion                                                  ation Sequence No. α001 tion                                          ______________________________________                                        IFN-A1                                                                              CTTGAAGGACAGACATG                                                                              6942     157-172                                                                              F                                         (SEQ ID NO:10)                                                               IFN-A2 CTGTCCTCCATGAGATG 6941 233-249 F                                        (SEQ ID NO:11)                                                               IFN-A3 GGTCATTCAGCTGCTGG 6940 339-355 R                                        (SEQ ID NO:12)                                                               IFN-A4 TCCTCCTTCATCAGGGG 6939 397-413 R                                        (SEQ ID NO:13)                                                               T3 ATTAACCCTCACTAAAG 13 Vector F                                               (SEQ ID NO:14)                                                               T7 TAATACGACTCACTATA 17 Vector R                                               (SEQ ID NO:15)                                                             ______________________________________                                    

All primers are shown from 5' to 3' orientation. The column designated"Direction" represents the direction of sequencing with respect to thesequence of the Hu-IFN-α: "F" represents forward: "R" - reverse.Oligodeoxynucleotides were synthesized on an Applied Biosystem DNAsynthesizer model 380B by the phosphoramidite method (83, 130).

A comparison of the protein sequence with other human interferon alphaspecies (Hu-IFN-α) (SEQ ID NO:2) demonstrates that Hu-IFN-α001 is mostclosely related to Hu-IFN-αJ (SEQ ID NO:3). That comparison isgraphically depicted in FIG. 2. A summary of the known Hu-IFN-αsequences has been previously reported (61). There are a total of sixamino acid changes compared to Hu-IFN-αJ. The data clearly demonstratethat this tumor derived Hu-IFN-α species is different from any otherknown Hu-IFN-α species previously reported. Furthermore, it would nothave been possible to predict this specific sequence as the number ofpossible proteins with alterations in these six positions is 20⁶ or64,000,000. One of the amino acid changes is in the signal peptidesequence; the remaining five alternations are in the mature protein. Itis also to be emphasized that the derived Hu-IFN-α species presentedhere is a natural interferon derived from tumor cells. It is not asynthetic construct prepared by simply mutating six positions.

Expression of the Hu-IFN-α001 gene was accomplished in two steps. Theplasmid pBS001 was digested with restriction endonuclease KpnI (5' endof Hu-IFN-α001 sequence). The KpnI ends were made blunt by incubationwith T4 DNA polymerase in the following reaction mixture: 1 μg of DNA;33 mM Tris acetate, pH 7.9; 66 mM potassium acetate; 10 mM magnesiumacetate; 0.5 mM dithiothreitol; 100 μg/ml BSA (bovine serum albumin); 2mM of each of the four dNTPs; 5 units of T4 DNA polymerase (UnitedStates Biochemical Corp.); total volume of 18 μl. Incubation wasperformed for 5 minutes at 37° C. to prepare the blunt ends. The plasmidDNA was then digested with XbaI (3' end of Hu-IFN-α001 sequence) torelease the insert containing the Hu-IFN-α001 sequence. The DNAfragments were then purified as described (86). The TGATG vector wasprepared by digestion with restriction endonuclease SacI, followed bypreparing blunt ends with T4 DNA polymerase as described above, and thendigested with restriction endonuclease XbaI. The fragment containing theHu-IFN-α001 insert was then ligated to the pTGATG expression vector(129). After ligation the DNA was transformed into competent E. coliDH5α cells. Colonies were analyzed by growing the cells as describedabove to isolate plasmid DNA. The plasmids were then digested withrestriction endonucleases EcoRI and XbaI to determine the size of theinsert. An expression vector for Hu-IFN-αJ was prepared as previouslydescribed for the expression plasmids for Hu-IFN-αB2 and Hu-IFN-αA/D(131).

The nucleotide sequences encoding Hu-IFN-αJ and Hu-IFN-α001 contain anAlwNI site in identical positions of the sequence (FIG. 3); and, asillustrated in FIG. 3, which shows the structure of the plasmidpHu-IFN-α001 containing the expression vector for Hu-IFN-α001, there isa second AlwNI site in the vector itself.

In addition, because the AlwNI recognition sites (CAGNNN0CTG) (SEQ IDNO:9) have three unspecified nucleotides (NNN) in the 3' overhang, thereligations are specific and asymmetric. Accordingly, pTGATG vectors(129) encoding Hu-IFN-αJ and Hu-IFN-α001 were digested with restrictionendonuclease AlwNI to isolate the large vector and Hu-IFN-α001 (3' endfragments, respectively. The Hu-IFN-α001 fragment was then ligated intothe vector fragment from plasmid pHu-IFN-αJ to yield the E. coliexpression vector pHu-IFN-α001, as shown in FIG. 3, which wastransfected into competent E. coli DH5α) cells (86).

Plasmid pHu-IFN-α001 is deposited with the American Type CultureCollection (ATCC) at 12301 Parklawn Drive, Rockville, Md. 20852:Accession number: 69640; Deposit date Jun. 7, 1994; and designated asplasmid pHu-IFN-α001 (E. coli DH5α pHu-IFN-α001 as the host vectorsystem).

The E. coli (DH5α) cells containing the expression vector pHu-IFN-α001were grown in 875 ml of Medium A overnight at 30° C. in one 2 literflask with rotary shaking. Medium A consists of KH₂ PO₄ (4.5 g/L). Na₂HPO₄.7H₂ O (18.9 g/L). NH₄ Cl (1.5 g/L), NaCl (0.75 g/L), glucose (15g/L), casamino acids (7.5 g/L). MgSO₄.7H₂ O (0.369 g/L), thiaminehydrochloride (0.0015 g/L), leucine (0.04 g/L), proline (0.04 g/L) andampicillin (0.05 g/L) adjusted to pH 7.4. The overnight culture was usedto inoculate 22.5 liters of Medium A in a fermentor. The E. colicontaining the expression vector were grown at 30° C. until the A₅₅₀reached 7.0 at which time the temperature was raised to 42° C. The cellswere harvested 3 hrs after temperature induction at 42° C. bycentrifugation and cell pellets divided into 50 g portions prior tofreezing at -80° C. The cells were stored at -80° C. until used forpreparation of interferon.

PURIFICATION OF Hu-IFN-α001

For purification of Hu-IFN-α001, frozen E. coli cell paste was thawed bysuspension in 10 volumes of Buffer A (50 mM Tris.HCl, pH 8.0, 50 mMNaCl, 10 mM EDTA, 0.1 mM PMSF, phenylmethylsufonylfluoride). After theaddition of egg white lysozyme (0.2 mg/ml) the suspension was sonicatedfour times with 30 second bursts while kept in an ice bath, thenincubated at 23° C. overnight while stirring vigorously to eliminateviscosity contributed by DNA. The suspension was centrifuged for 20minutes at 12,000 rpm at 4° C. The pellet was resuspended again in 10volumes of Buffer A with 1% Triton X-100, 50 mM EDTA and 0.5 M NaCl andincubated for at least 2 hours (2-16 hrs) at room temperature withshaking and then centrifuged for 20 min at 12,000 rmp at 4° C. Onceagain, the pellet was resuspended in 5 volumes of Buffer A with 0.5 MNaCl and incubated for 60 min at room temperature with shaking and thencentrifuged for 20 min at 12,000 rpm at 4° C.; the supernatant wasdiscarded. The pellet was dispersed in 2 volumes of Buffer A in thepresence of a mixture of oxidized/reduced forms of glutathione (0.2mM/2.0 mM) and solid guanidine.HCl (2.5 times bacterial weight) wasadded and the solution was stirred at room temperature for 7 hours.After this, the mixture was diluted tenfold with Buffer A and allowed tostand overnight. Renaturation of the interferon was carried out by veryslow addition of 7 M guanidine.HCl to 0.7 M. The refolding ofHu-IFN-α001 in solution takes more than 15 hours. Since Hu-IFN-α001contains two disulfide bonds, this step involves slow oxidation of theprotein during dilution from guanidine-containing solution. Thensuspension was then centrifuged to remove debris. Solid (NH₄)₂ SO₄ wasadded to the supernatant to a final concentration 1 M, and the solution,after clarification by centrifugation, was loaded at 5 ml/min onto acolumn (Pharmacia XK 2620=18-1000-72) packed with 100 ml of the sorbentPhenyl-Toyopearl 650 S (20-50 μm) (Supelco, #8-14477: 100 g), previouslyequilibrated with 3-4 column volumes of Buffer B (50 mM Tris.HCl, pH7.4, 0.5 M guanidine.HCl and 1 M (NH₄)₂ SO₄. The column effluent wasmonitored at 280 nm. After loading, the column was washed with Buffer Buntil the A₂₈₀ of the effluent returned to near baseline level and thenwas eluted sequentially with 2-3 column volumes of Buffer C (50 mMTris.HCl, 0.5 M guanidine.HCl, 0.6 M (NH₄)₂ SO₄) with which theHu-IFN-α001 was eluted. Peak fractions showing maximum bands ofHu-IFN-α001 on SDS-polyacrylamide gel electrophoresis were pooled. ThePhenyl-Toyopearl column was regenerated in situ with 100 ml 0.5 M NaOHand 1 M NaCl solution; and was stored in 0.01% sodium azide. Fractionswith Hu-IFN-001 as measured by antiviral activity and/or gelelectrophoresis were pooled and concentrated 10-fold with an AmiconCentriprep 10 concentrator. The solution was then diluted 3-fold withBuffer D (20 mM Tris.HCl, pH 8.0, 5% glycerol) and was loaded onto aFPLC monoQ HR 10/10 ion exchange column (Pharmacia #17-0556-01)equilibrated with Buffer D. The column was washed with about 10 ml ofBuffer D until the A₂₈₀ reach baseline. Elution of Hu-IFN-α001 wasaccomplished with a linear gradient of Buffer D and Buffer E (Buffer Dplus 1 M NaCl) at a flow rate of 1.5 ml/min from 0 to 100% Buffer E over3 hours. The Hu-IFN-α001 was eluted at 0.15 M NaCl in a single peak. Thefractions were pooled, analyzed by sodium dodecylsulfate (SDS)polyacrylamide gel electrophoresis and assayed for antiviral activity.From 6 g of bacterial pellet (wet weight), about 8-10 mg of purifiedHu-IFN-α001 was obtained.

The purified protein was mixed with 15 μl of SDS sample buffer (0.5 MTris.HCl, pH 6.8, 1% (v/v) β-mercaptoethanol, 1% (w/v) sodiumdodecylsulfate (SDS), 12% (v/v) glycerol, 2 mM ethylenediaminetetraceticacid (EDTA), bromophenol blue) in a total volume of 35 μl. The solutionwas boiled for two minutes after which 25 μl was loaded onto a 12.5%polyacrylamide gel with a 4% polyacrylamide stacking gel. The separatinggel was buffered in 0.3 M Tris.HCl, 0.08% SDS, 2 mM EDTA, pH 8.8. Thestacking gel was in 0.065 M Tris.HCl, pH 6.8, and 0.05% SDS. The chamberbuffer was 25 mM Tris.HCl, 0.1% SDS, 0.2 M glycine. Electrophoresis wascarried out for 1 hour at 150 V, 20 mA in the BioRad miniprotein IIapparatus (132). The gel as stained with Coomassie Blue R-250 (2.4% w/v.Coomassie Blue in 45% methanol, 9%, v/v, acetic acid) for 1 hour at roomtemperature; and destained in 8% acetic acid. From SDS-polyacrylamidegel electrophoresis it was apparent that the purified Hu-IFN-α001migrated with a M of 20,000 as shown in FIG. 4. As indicated in thatfigure, Hu-IFN-α001 was placed in lanes 1, 2 and 3 in amounts of 3 μg,1.5 μg and 0.75 μg, respectively. The columns labeled M represent themolecular weight markers with the values in kilodaltons given to theleft of each respective molecular weight marker. As can be seen, theHu-IFN-α001 exhibited a slightly slower mobility than Hu-IFN-αJ onSDS-polyacrylamide gel electrophoresis (SDS PAGE, ref. 132).

Antiviral activity of Hu-IFN-α001 was assayed on bovine MDBK and humanFS7 cells with vesicular stomatitis virus (VSV) (Table 5) as describedpreviously (133). The antiviral units were determined with respect tothe human IFN-αA international standard Gxa01-901-535. There wasapproximately equal antiviral activity on human and bovine cells (Table5) as is seen with many Hu-IFN-α species (17,27,30,100,103,134).

                  TABLE 5                                                         ______________________________________                                        Antiviral Assay of Interferon                                                         Interferon Titer (units/ml)                                                                     Ratio                                               Sample  FS-7 Cells  MDBK Cells                                                                              (FS-7/MDBK)                                     ______________________________________                                        α001                                                                            1 × 10.sup.8                                                                        1 × 10.sup.8                                                                      1.0                                             ______________________________________                                    

The interferon titer is given in units/mg as described(10-12,99,100,133,135) with respect to the international standard forhuman interferon alpha A Gxa01-901-535 from the National Institutes ofHealth. Vesicular stomatitis virus (VSV) was used as the challenge viruswith human FS-7 and bovine MDBK cells. The ratio of the antiviralactivity of the interferon on FS-7 to that on MDBK cells is given in thelast column. The samples of Hu-IFN-α001 were prepared as described inthe text. Protein was determined by the method of Bradford (136).

Herein has been described an entire new class of molecules designated assuper proteins, proteins not present in normal cells, but present in thecells in various diseased states and a method for identifying, producingand expressing such molecules. Although the present embodiment of theinvention has been described in detail, it should be understood thatvarious changes, alterations and substitutions can be made thereinwithout departing from the spirit and scope of the invention as definedby the appended claims.

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88. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) "DNA Sequencingwith Chain Terminating Inhibitors," Proc. Natl. Acad. Sci. USA 74,5463-5467.

89. Tabor, S., and Richardson, C. C. (1987) "DNA Sequencing Analysiswith a Modified Bacteriophage T7 DNA Polymerase," Proc. Natl. Acad. Sci.USA 84, 4767-4771.

90. Kawasaki, E. S., and Wang, A. M. (1989) "Detection of GeneExpression," In: PCR Technology: Principles and Applications of DNAAmplification (Erlich, H. A. ed.) Stockton Press, Inc., New York, N.Y.,pp 89-97.

91. McCandliss, R., Sloma, A., and Pestka, S. (1981) "Isolation andCell-free Translation of Human Interferon mRNA from Fibroblasts andLeukocytes," in Methods in Enzymology, Vol. 79 (S. Pestka, ed.),Academic Press, New York, 51-59.

92. Chomczynski, P., and Sacchi, N. (1987) "Single Step Method of RNAIsolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction,"Anal. Bioch. 162, 156-159.

93. Pellicer, A., Wigler, M., and Axel, R. (1978) "The Transfer andStable Integration of the HSV Thymidine Kinase Gene into Mouse Cells,"Cell 14, 133-14.

94. Gross-Bellard, M., Oudet, P., and Chambon, P. (1973) "Isolation ofHigh-Molecular-Weight DNA from Mammalian Cells," Eur. J. Biochem. 36,32-38.

95. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Hom, G. T.,Erlich, H. A., and Arnheim, N. (1985) "Enzymatic Amplification ofBeta-Globin Genomic Sequences and Restriction Site Analysis forDiagnosis of Sickle Cell Anemia," Science 230, 1350-1354.

96. Keohavong, P., and Thilly, W. G. (1989) "Fidelity of DNA Polymerasesin DNA Amplification," Proc. Natl. Acad. Sci. USA 86, 9253-9257.

97. Hotta, K., Collier, K. J., and Pestka, S. (1986) "Detection of aSingle Base Substitution Between Human Leukocyte Interferon αA and α2Genes with Octadecyl Deoxyoligonucleotide Probes," in Methods inEnzymology (S. Pestka, ed.), Academic Press, New York 119, 481-485.

98. Hotta, K., Monohan, J., Collier, K. J., and Pestka, S. (1988)"Detection of Human Leukocyte Interferon αA and α2 Genes in Genomic DNAsby the Use of Deoxyoctadecyloligonucleotide Probes," J. Interferon Res.8, 51-60.

99. Pestka, S., and Baron, S. (1981) "Definition and Classification ofthe Interferons," in Methods in Enzymology (S. Pestka, ed.), AcademicPress, New York, 78, 3-14.

100. Pestka, S. (1986) "Interferon Standards and General Abbreviations,"in Methods in Enzymology (S. Pestka, ed.), Academic Press, New York,119, 14-23.

101. Evinger, M., Maeda, S., and Pestka, S. (1981) "Recombinant HumanLeukocyte Interferon Produced in Bacteria Has AntiproliferativeActivity," J. Biol. Chem. 256, 2113-2114.

102. Herberman, R. B., Ortaldo, J. R., Mantovani, A., Hobbs, D. S.,Kung, H.-F., and Pestka, S. (1982) "Effect of Human RecombinantInterferon on Cytotoxic Activity of Natural Killer (NK) Cells andMonocytes," Cell Immunol. 67, 160-167.

103. Rehberg, E., Kelder, B., Hoal, E. G., and Pestka, S. (1982)"Specific Molecular Activities of Recombinant and Hybrid LeukocyteInterferons," J. Biol. Chem. 257, 11497-11502.

104. Jones, C. M., Varesio, L., Herberman, R. B., and Pestka, S. (1982)"Interferon Activates Macrophages to Produce Plasminogen Activator," J.Interferon Res. 2, 377-386.

105. Grant, S., Bhalla, K., Weinstein, I. B., Pestka, S., and Fisher, P.B (1982) "Differential Effect of Recombinant Human Leukocyte Interferonon Human Leukemic and Normal Myeloid Progenitor Cells," Biochem.Biophys. Res. Commun. 108, 1048-1055.

106. Ortaldo, J. R., Herberman, R. B., and Pestka, S. (1982)"Augmentation of Human Natural Killer Cells with Human Leukocyte andHuman Recombinant Leukocyte Interferon," in NK Cells and Other NaturalEffector Cells (R. B. Herberman, ed.), Academic Press, New York,1279-1283.

107. Ortaldo, J. R., Mason, A., Rehberg, E., Moschera, J., Kelder, B.,Pestka, S., and Herberman, R. B. (1983) "Effects of Recombinant andHybrid Recombinant Human Leukocyte Interferons on Cytotoxic Activity ofNatural Killer Cells," J. Biol. Chem. 258, 15011-15015.

108. Fisher, P., Miranda, A. F., Babiss, L. E., Pestka, S., andWeinstein, I. B. (1983) "Opposing Effects of Interferon Produced inBacteria and of Tumor Promoters on Myogenesis in Human MyoblastCultures," Proc. Natl. Acad. Sci. U.S.A. 80, 2961-2965.

109. Sen, G. C., Herz, R. E., Davatelis, V., and Pestka, S. (1984)"Antiviral and Protein-Inducing Activities of Recombinant HumanLeukocyte Interferons and Their Hybrids," J. Virol. 50, 445-450.

110. Giacomini, P., Aguzzi, A., Pestka, S., Fisher, P. B., and Ferrone,S. (1984) "Modulation by Recombinant DNA Leukocyte (α) and Fibroblast(β) Interferons of the Expression and Shedding of HLA and TumorAssociated Antigens by Melanoma Cells," J. Immunol. 133, 1649-1655.

111. Greiner, J. W., Hand, P. H., Noguchi, P., Fisher, P. B., Pestka,S., and Schlom, J. (1984) "Enhanced Expression of SurfaceTumor-Associated Antigens on Human Breast and Colon Tumor Cells AfterRecombinant Human Leukocyte α-Interferon Treatment," Cancer Res. 44,3208-3214.

112. Fisher, P. B., Prignoli, D. R., Hermo, H., Jr., Weinstein, I. B.,and Pestka, S. (1985) "Effects of Combined Treatment with Interferon andMezerein on Melanogenesis and Growth in Human Melanoma Cells," J.Interferon Res. 5, 11-22.

113. Grant, S., Bhalla, K., Weinstein, I. B., Pestka, S., Mileno, M. D.,and Fisher, P. B. (1985) "Recombinant Human Interferon SensitizesResistant Myeloid Leukemic Cells to Induction of TerminalDifferentiation," Biochem. Biophys. Res. Commun. 130, 379-388.

114. Guadagni, F., Schlom, J., Johnston, W. W., Szpak, C. Z., Goldstein,D., Smalley, R., Simpson, J. F., Borden, E. C., Pestka, S., and Greiner,J. W., (1989) "Selective Interferon-Induced Enhancement ofTumor-Associated Antigens on a Spectrum of Freshly Isolated HumanAdenocarcinoma Cells," J. Natl. Cancer Inst. 81, 502-512.

115. Sperber, S. J., Gocke, D. J., Haberzettl, C., Kuk, R., Schwartz,B., and Pestka, S. (1992) "Anti-HIV-1 Activity of Recombinant and HybridSpecies of Interferon Alpha," J. Interferon Res. 12, 363-368.

116. Huber, C., Flener, R., and Gastl, G. (1985) "Interferon-Alpha-2c inthe Treatment of Advanced Hairy Cell Leukemia," Oncology 42, Suppl 1,7-9.

117. Foon, K. A., Maluish, A. E., Abrams, P. G., Wrightington, S.,Stevenson, H. C., Alarif, A., Fer, M. F., Overton, W. R., and Poole, M.(1986) "Recombinant Leukocyte A Interferon Therapy for Advanced HairyCell Leukemia," Am. J. Med. 80, 351-356.

118. Steis, R. G., Smith, J. W. II, Urba, W. J., Clark, J. W., Itri, L.M., Evans, L. M., Schoenberge, C., and Longo, D. L. (1988) "Resistanceto Recombinant Interferon Alfa-2 in Hairy Cell Leukemia Associated withNeutralizing Anti-interferon Antibodies," New Engl. J. Med. 318,1409-1413.

119. Von Wussow, P., Freund, M., Hartmann, F., Deidrich, H., Poliwoda,H., and Deicher, H. (1987) "Anti Interferon Antibodies: Pharmokineticsand Clinical Significance," J. Interferon Res. 7, 680.

120. Freund, M., Von Wussow, P., Diedrich, H., Eisert, R., Link, H.,Wilke, H., Buchholz, F., LeBlanc, S., Fonatsch, C., Deicher, H., andPoliwoda, H. (1989) "Recombinant Human Interferon (IFN) Alpha-2b inChronic Myelogenous Leukemia: Dose Dependency of Response and Frequencyof Neutralizing Anti-Interferon Antibodies," Br. J. Haematol. 72,350-356.

121. Itri, L. M., Campion, M., Dennin, R. A., Palleroni, A. V.,Gutterman, J. O., Groopman, J. E., and Trown, P. W. (1987) "Incidenceand Clinical Significance of Neutralizing Antibodies in PatientsReceiving Recombinant Interferon Alpha-1A by Intramuscular Injection,"Cancer 59, 668-674.

122. Moormeier, J. A., Westbrook, C. A., Ratain, M. J., and Golomb, H.M. (1989) "Interferon Alfa-2b Antibodies and Clinical Resistance in aPatient with Hairy Cell Leukemia," Leuk. Lymphoma 1, 43-45.

123. Quesada, J. R., Rios, A., Swanson, D. A., Trown, P., and Gutterman,J. U. (1985) "Antitumor Activity of Recombinant-derived Interferon Alphain Metastatic Renal Cell Carcinoma," J. Clin. Oncol. 3, 1522-1528.

124. Antonelli, G., Currenti, M., Turriziani, O., and Dianzani, F.(1991) "Neutralizing Antibodies to Interferon α: Relative Frequency inPatients Treated with Different Interferon Preparations," J. Infect.Dis. 163, 882-885.

125. Colamonici, O., Porterfield, B., and Diaz, M. O. (1991) "InterferonSensitivity of Human Leukemia Cell Lines With and Without Deletion ofthe Interferon Genes," J. Interferon Res. 11, S54.

126. Grander, D., Heyman, M., Brondum-Nielsen, K., Liu, Y., Lundgren,E., Soderhall, S., and Einhorn, S. (1992) "Interferon System in PrimaryAcute Lymphocytic Leukemia Cells With or Without Deletions of theα-/β-Interfron Genes," Blood 79, 2076-2083.

127. Nishimura, A., Morita, M., Nishimura, Y. and Sugino, Y. (1990) "ARapid and Highly Efficient Method for Preparation of CompetentEscherichia coli Cells," Nucleic Acids Research 18, 6169.

128. Lee, S. and Rashid, S. (1990) "A Simple Procedure for Maximum Yieldof High-quality Plasmid DNA," BioTechniques 9, 676-679.

129. Mashko, S. V., Veiko, V. P., Lapidus, A. L., Lebedeva, M. I.,Mochculsky, A. V., Schechter, I. I., Trukhan, M. E., Ratmanova, K. I.,Rebentish, B. A., Kaluzhsky, V. E. and Debabov, V. G. (1990) TGATGvector: a new expression system for cloned foreign genes in Escherichiacoli cells. Gene 88, 121-126.

130. Caruthers, M. H., Barone, A. D., Beaucage, S. L., Dodds, D. R.,Fisher, E. F., McBride, L. J., Matteucci, M., Stabinsky, Z., Tang, J. Y.(1987) "Chemical Synthesis of Deoxyoligonucleotides by thePhosphoramidite Method," Methods in Enzymology 154, 287-313.

131. Wang, P., Izotova, L., Mariano, T. M., Donnelly, R. J. and Pestka,S. (1994) "Construction and Activity of Phosphorylatable HumanInterferon-αB2 and Interferon-αA/D." J. Interferon Res. 14, 41-46.

132. Laemmli, U. K. (1990) Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London) 227: 680-685

133. Familletti, P. C., Rubinstein, S., and Pestka, S. (1981) "AConvenient and Rapid Cytopathic Effect Inhibition Assay for Interferon,"in Methods in Enzymology, Vol. 78 (S. Pestka, ed.), Academic Press, NewYork, 387-394.

134. Staehelin, T., Hobbs, D. S., Kung, H.-F., Lai, C.-Y., and Pestka,S. (1981) "Purification and Characterization of Recombinant HumanLeukocyte Interferon (IFLrA) with Monoclonal Antibodies," J. Biol. Chem.256, 9750-9754.

135. Pestka, S. (1981) "Standard Media and General Abbreviations," inMethods in Enzymology, Vol. 78 (S. Pestka, ed.), Academic Press, NewYork, 22-25.

136. Bradford, M. M. (1976) "A Rapid and Sensitive Method for thequantitation of Microgram quantities of Protein Utilizing the Principleof Protein-dye Binding," Anal. Biochem. 72, 248-254.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 16                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 570 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Homo sapi - #ens                                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..570                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATG GCC TTG TCC TTT TCT TTA CTG ATG GTC GT - #G CTG GTA CTC AGC        TAC       48                                                                    Met Ala Leu Ser Phe Ser Leu Leu Met Val Va - #l Leu Val Leu Ser Tyr            1               5 - #                 10 - #                 15              - - AAA TCC ATC TGC TCT CTG GGC TGT GAT CTG CC - #T CAG ACC CAC AGC CTG           96                                                                       Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pr - #o Gln Thr His Ser Leu                        20     - #             25     - #             30                  - - CGT AAT AGG AGG GCC TTG ATA CTC CTG GCA CA - #A ATG GGA AGA ATC TCT          144                                                                       Arg Asn Arg Arg Ala Leu Ile Leu Leu Ala Gl - #n Met Gly Arg Ile Ser                    35         - #         40         - #         45                      - - CCT TTC TCC TGC TTG AAG GAC AGA CAT GAA TT - #C AGA TTC CCA GAG GAG          192                                                                       Pro Phe Ser Cys Leu Lys Asp Arg His Glu Ph - #e Arg Phe Pro Glu Glu                50             - #     55             - #     60                          - - GAG TTT GAT GGC CAC CAG TTC CAG AAG ACT CA - #A GCC ATC TCT GTC CTC          240                                                                       Glu Phe Asp Gly His Gln Phe Gln Lys Thr Gl - #n Ala Ile Ser Val Leu            65                 - # 70                 - # 75                 - # 80       - - CAT GAG ATG ATC CAG CAG ACC TTC AAT CTC TT - #C AGC ACA GAG GAC TCA          288                                                                       His Glu Met Ile Gln Gln Thr Phe Asn Leu Ph - #e Ser Thr Glu Asp Ser                            85 - #                 90 - #                 95              - - TCT GCT GCT TGG GAA CAG AGC CTC CTA GAA AA - #A TTT TCC ACT GAA CTT          336                                                                       Ser Ala Ala Trp Glu Gln Ser Leu Leu Glu Ly - #s Phe Ser Thr Glu Leu                       100      - #           105      - #           110                  - - TAC CAG CAA CTG AAT GAC CTG GAA GCA TGT GT - #G ATA CAG GAG GTT GGG          384                                                                       Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Va - #l Ile Gln Glu Val Gly                   115          - #       120          - #       125                      - - GTG GAA GAG ACT CCC CTG ATG AAT GAG GAC TC - #C ATC CTG GCT GTG AGG          432                                                                       Val Glu Glu Thr Pro Leu Met Asn Glu Asp Se - #r Ile Leu Ala Val Arg               130              - #   135              - #   140                          - - AAA TAC TTC CAA AGA ATC ACT CTT TAT CTA AC - #A GAG AAG AAA TAC AGC          480                                                                       Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Th - #r Glu Lys Lys Tyr Ser           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - CCT TGT GCC TGG GAG GTT GTC AGA GCA GAA AT - #C ATG AGA TCC CTC        TCG      528                                                                    Pro Cys Ala Trp Glu Val Val Arg Ala Glu Il - #e Met Arg Ser Leu Ser                          165  - #               170  - #               175              - - TTT TCA ACA AAC TTG CAA AAA AGA TTA AGG AG - #G AAG GAT TGA                 - # 570                                                                    Phe Ser Thr Asn Leu Gln Lys Arg Leu Arg Ar - #g Lys Asp                                   180      - #           185      - #           190                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 189 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Ala Leu Ser Phe Ser Leu Leu Met Val Va - #l Leu Val Leu Ser Tyr        1               5 - #                 10 - #                 15              - - Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pr - #o Gln Thr His Ser Leu                   20     - #             25     - #             30                  - - Arg Asn Arg Arg Ala Leu Ile Leu Leu Ala Gl - #n Met Gly Arg Ile Ser               35         - #         40         - #         45                      - - Pro Phe Ser Cys Leu Lys Asp Arg His Glu Ph - #e Arg Phe Pro Glu Glu           50             - #     55             - #     60                          - - Glu Phe Asp Gly His Gln Phe Gln Lys Thr Gl - #n Ala Ile Ser Val Leu       65                 - # 70                 - # 75                 - # 80       - - His Glu Met Ile Gln Gln Thr Phe Asn Leu Ph - #e Ser Thr Glu Asp Ser                       85 - #                 90 - #                 95              - - Ser Ala Ala Trp Glu Gln Ser Leu Leu Glu Ly - #s Phe Ser Thr Glu Leu                  100      - #           105      - #           110                  - - Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Va - #l Ile Gln Glu Val Gly              115          - #       120          - #       125                      - - Val Glu Glu Thr Pro Leu Met Asn Glu Asp Se - #r Ile Leu Ala Val Arg          130              - #   135              - #   140                          - - Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Th - #r Glu Lys Lys Tyr Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Cys Ala Trp Glu Val Val Arg Ala Glu Il - #e Met Arg Ser Leu        Ser                                                                                             165  - #               170  - #               175             - - Phe Ser Thr Asn Leu Gln Lys Arg Leu Arg Ar - #g Lys Asp                              180      - #           185                                         - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 189 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Homo sapi - #ens                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Met Ala Arg Ser Phe Ser Leu Leu Met Val Va - #l Leu Val Leu Ser Tyr      1               5   - #                10  - #                15               - - Lys Ser Ile Cys Ser Leu Gly Cys Asp Leu Pr - #o Gln Thr His Ser Leu                  20      - #            25      - #            30                   - - Arg Asn Arg Arg Ala Leu Ile Leu Leu Ala Gl - #n Met Gly Arg Ile Ser              35          - #        40          - #        45                       - - Pro Phe Ser Cys Leu Lys Asp Arg His Glu Ph - #e Arg Phe Pro Glu Glu          50              - #    55              - #    60                           - - Glu Phe Asp Gly His Gln Phe Gln Lys Thr Gl - #n Ala Ile Ser Val Leu      65                  - #70                  - #75                  - #80        - - His Glu Met Ile Gln Gln Thr Phe Asn Leu Ph - #e Ser Thr Glu Asp Ser                      85  - #                90  - #                95               - - Ser Ala Ala Trp Glu Gln Ser Leu Leu Glu Ly - #s Phe Ser Thr Glu Leu                  100      - #           105      - #           110                  - - Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys Va - #l Ile Gln Glu Val Gly              115          - #       120          - #       125                      - - Val Glu Glu Thr Pro Leu Met Asn Glu Asp Ph - #e Ile Leu Ala Val Arg          130              - #   135              - #   140                          - - Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Me - #t Glu Lys Lys Tyr Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Cys Ala Trp Glu Val Val Arg Ala Glu Il - #e Met Arg Ser Phe        Ser                                                                                             165  - #               170  - #               175             - - Phe Ser Thr Asn Leu Lys Lys Gly Leu Arg Ar - #g Lys Asp                              180      - #           185                                         - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                                        (A) DESCRIPTION: Primer - #I                                         - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TGGGCTGTGA TCTGCCTC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                                        (A) DESCRIPTION: Primer - #II                                        - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - CATGATTTCT GCTCTGACAA CC           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                                        (A) DESCRIPTION: Primer - #III                                       - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - AACCCACAGC CTGGGTAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GCGGGCCCCA ATGGCCYTGY CCTTT          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GCTCTAGAAY TCATGAAAGY GTGA          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 base p - #airs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                                        (A) DESCRIPTION: AlwNI - #restriction endonuclease recognition                     site used - # to splice the Hu-IFN-`001 wherein N             represents                                                                                     any nucle - #otide                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CAGNNNCTG                - #                  - #                      - #          9                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                                        (A) DESCRIPTION: Primer - #used in sequencing of Hu-IFN-`            - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - CTTGAAGGAC AGACATG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                                        (A) DESCRIPTION: Primer - #used in sequencing of Hu-IFN-`            - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - CTGTCCTCCA TGAGATG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                                        (A) DESCRIPTION: Primer - #used in sequencing of Hu-IFN-`            - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GGTCATTCAG CTGCTGG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                                        (A) DESCRIPTION: Primer - #used in sequencing of Hu-IFN-`            - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - TCCTCCTTCA TCAGGGG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                                        (A) DESCRIPTION: Primer - #used in sequencing of Hu-IFN-`            - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - ATTAACCCTC ACTAAAG             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                                        (A) DESCRIPTION: Primer - #used in sequencing of Hu-IFN-`            - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - TAATACGACT CACTATA             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 166 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -    (iii) HYPOTHETICAL: NO                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: Hu-IFN-alpha001                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Cys Asp Leu Pro Gln Thr His Ser Leu Arg As - #n Arg Arg Ala Leu Ile      1               5   - #                10  - #                15               - - Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Ph - #e Ser Cys Leu Lys Asp                  20      - #            25      - #            30                   - - Arg His Glu Phe Arg Phe Pro Glu Glu Glu Ph - #e Asp Gly His Gln Phe              35          - #        40          - #        45                       - - Gln Lys Thr Gln Ala Ile Ser Val Leu His Gl - #u Met Ile Gln Gln Thr          50              - #    55              - #    60                           - - Phe Asn Leu Phe Ser Thr Glu Asp Ser Ser Al - #a Ala Trp Glu Gln Ser      65                  - #70                  - #75                  - #80        - - Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gl - #n Gln Leu Asn Asp Leu                      85  - #                90  - #                95               - - Glu Ala Cys Val Ile Gln Glu Val Gly Val Gl - #u Glu Thr Pro Leu Met                  100      - #           105      - #           110                  - - Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Ty - #r Phe Gln Arg Ile Thr              115          - #       120          - #       125                      - - Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cy - #s Ala Trp Glu Val Val          130              - #   135              - #   140                          - - Arg Ala Glu Ile Met Arg Ser Leu Ser Phe Se - #r Thr Asn Leu Gln Lys      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Leu Arg Arg Lys Asp                                                                  165                                                         __________________________________________________________________________

I claim the following:
 1. A method of identifying a modified polypeptideencoded by a gene that has been mutated, wherein the polypeptide is asecreted protein selected from the group consisting of cytokines,lymphokines, growth factors, adhesion molecules, enzymes, clottingfactors, peptide hormones, and polypeptide hormones, and wherein thepolypeptide is not encoded by a oncogene or tumor suppressor gene, andwherein the mutation occurs during the pathological process of tumorformation, comprising the steps of:selecting tumor, cancer, or culturedcell line cells from which a specific nucleotide sequence encoded apolypeptide from the group consisting of cytokines, lymphokines, growthfactors, adhesion molecules, enzymes, clotting factors, peptidehormones, and polypeptide hormones, may be obtained; cloning at least aportion of a nucleotide sequence from the tumor, cancer, or culturedcell line cells that encodes the polypeptide; sequencing the clonednucleotide sequence and predicting therefrom the polypeptide amino acidsequence; and comparing the amino acid sequence of the polypeptide withan amino acid sequence for the polypeptide from non-diseased cells todetermine whether the polypeptide has been mutated compared to thepolypeptide from non-diseased cells.
 2. The method of claim 1 whereinsaid selected tumor, cancer, or cultured cell line cells are derivedfrom hemopoietic cells.
 3. The method of claim 1 wherein said selectedtumor, cancer, or cultured cell line cells are derived from leukemicleukocytes.
 4. The method of claim 1 wherein said selected tumor,cancer, or cultured cell line cells are derived from human malignancies.5. The method of claim 1 further comprising a step of expressing saidmodified polypeptide by expressing a DNA sequence coding for saidmodified polypeptide in a host cell.
 6. The method of claim 1 whereinsaid cloning step comprises cloning said nucleotide sequence byinsertion in an appropriate plasmid, an appropriate host beingthereafter transformed with said plasmid.